PROJECT SUMMARY Advances in tissue engineering have reinvigorated efforts to develop an in vitro articular tissue graft for the temporomandibular joint (TMJ). Importantly, recapitulating the native biomolecular components is critical to supporting compressive and tensile loading of the articular disc. Tissue engineers must conduct careful selection of cell sources in addition to developing a comprehensive understanding of the native biomolecules present in the target tissue. A relative lack of developmental studies addressing the genetic, molecular, and tissue level properties of the TMJ has created an unclear picture of characteristics tissue engineers and regenerative biomedical experts need to reproduce. Most often, outcomes for these studies utilize the same parameters used for regenerating hyaline cartilage. While there is a substantial and valuable body of knowledge available for regenerating hyaline cartilage, the key components are very different for the articular disc of the TMJ and fall short of addressing unique gliding, rotating, compressive and most notably tensile forces on the articular disc. This project addresses the challenge of recapitulating necessary biomolecular components that functionally support mechanical properties of articular disc tissue. It is based on preliminary work in which we have discovered a novel cell source, tissue-specific progenitor cells from the disc itself, for application in regenerative tissue engineering approaches. Furthermore, we have produced data suggesting a very different perspective needs to be drawn on the developmental origins of the articular disc, which affects the targets the tissue engineering approach will have. In figure 4 of this proposal's research strategy, one can see a the TMJ of a scleraxisGFP reporter mouse. The GFP detected indicates expression of this key tendon marker, indicating the articular disc is in fact a continuous structure with tendon characteristics. This finding, coupled with other supporting evidence has led me to propose the target structure for tissue engineering should not be the hyaline cartilaginous phenotype seen in published studies from other groups, but rather a phenotype that includes both tendon proteins and those proteins observed in compressively loaded tendons, namely cartilage-associated proteins. This project proposes to use whole-mount immunostaining, tissue optical clearing, state-of-the art microscopy techniques, and quantitative biomolecular techniques to further characterize the components of the articular disc. This will be accompanied by an investigation into the role of mechanical forces and growth factors that induce expression of these components in our tissue engineered constructs. If successful, the project will provide a solid basis for larger basic science and translational studies into TMJ disc replacement therapies. !